CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway

Abstract

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.

mTOR inhibition via Rapamycin treatment partially reverts the deficit in energy metabolism caused by FH loss in RPE cells

“…For instance, small drusen (<63µm in diameter, now commonly called drupelets) can be found in the non-AMD-related aging retina [47]. Similarily, slight, but constantly elevated mTOR activity together with an elevation of proinflammatory or stress related factors [31,33,48] may accelerate age-associated processes that push the development of AMD. Previous studies have shown that FH dysregulation in hTERT-RPE1 cells leads to increased vulnerability towards oxidative stress [32].…”

“…The fact that the exogenous supplementation of recombinant FH was not able to reverse the defects in cellular metabolism elicited by FH knockdown in RPE cells [32,33] points towards a non-canonical, intracellular, role of endogenous FH. Interestingly, the non-canonical intracellular role of FH has been recently described in other cell types, showing a similar phenotypes as RPE cells.…”

“…Protein expression was analyzed in both cell lysates and cell supernatants as previousely described [33]. Briefly, after debris removal, cell culture supernatants were precipitated with ice-cold acetone and cell lysates were prepared in Pierce IP Lysis Buffer, containing Halt Protease and Phosphatase Inhibitor (Thermo Fisher Scientific, Boston, MA, USA).…”

“…Equal protein amounts of cell lysates or equal volumes of cell supernatants were prepared in NuPAGE LDS Sample Buffer (Thermo Fisher Scientific, Boston, MA, USA), containing reducing agent (Thermo Fisher Scientific, Boston, MA, USA) and analyzed on Novex 8-16% Tris-Glycine gels (Invitrogen, Waltham, MA, USA). Subsequently, proteins were transferred onto PVDF membranes (Roche, Basel, Switzerland), and Western blot detection was carried out as previously described [32,33], using the primary antibodies listed in Table 2. Pictures were acquired with a FusionFX imaging system (Vilber Lourmat, Collégien, France), and the intensity density of individual bands was quantified using ImageJ (Version 1.53a).…”

“…In addition, several in vitro studies suggest that FH holds additional functions in RPE cells, like regulation of cellular energy metabolism, which are affected by the AMD-associated variant [31] or by reduced intracellular FH levels and activity [32]. In particular, in our previous studies we assessed the impact of reduced levels of functional FH in RPE cells and found that reduced FH levels are associated with increased inflammatory signaling via the NF-κB pathway [33] and profound metabolic disturbances along with mitochondrial damage [31,32]. The exogenous supplementation of recombinant FH was not able to reverse those effects, pointing towards a non-canonical role of endogenous FH.…”